Hb Nivaria: A New Hemoglobin Variant with a Shortened α-Globin Chain [α139(HC1)Lys→Stop; HBA1: c.418A>T].

We report a novel hemoglobin (Hb) variant found in a Spanish individual from Santa Cruz de Tenerife, the Canary Islands in Spain. The proband was a 39-year-old male. High performance liquid chromatography (HPLC) displayed an unknown peak (19.3%) at a retention time of 1.3 min. eluting before Hb A0. Capillary zone electrophoresis (CZE) showed an abnormal peak (20.0%) in zone 12. Direct DNA sequencing of the α-globin genes revealed heterozygosity for a nonsense mutation at codon 139 (AAA>TAA), causing a lysine to stop codon substitution [α139(HC1)Lys→Stop; HBA1: c.418A>T]. We decided to name the variant Hb Nivaria (Tenerife) for the place of birth and residence of the proband.

C>A substitution in NT 46 of the 3' UTR region (the α complex protected region) of the alpha-1 globin gene: a non-deletional mutation or polymorphism?

AIMS: Untranslated regions (UTRs) play an important role in post-transcriptional regulation of gene expression, including by modulating messenger RNA (mRNA) transport out of the nucleus, translation efficiency, subcellular localisation and stability. Any mutation in this region could alter the stability of mRNA and thereby affect protein synthesis. We analysed if a mutation located in the α complex protected region of the α1 globin gene could cause non-deletional α-thalassaemia by affecting post-transcriptional stability (mRNA stability). METHODS: A total of 14 patients without anaemia, normal or slight microcytosis and hypochromia (medium concentration haemoglobin [MCH] <27 pg) were studied. Haemoglobin subtypes were screened using capillary zone electrophoresis and ion-exchange high-performance liquid chromatography (VARIANT II ß-Thalassaemia Short Program). The most common α-globin mutations were identified by multiplex PCR (Alpha-Globin StripAssay kit) and the molecular characterisation by automatic sequencing of alpha globin genes. RESULTS: All of them shown a novel transversion mutation in nt 778 (C>A), which is located in the 3' UTR in the α complex protected region [HBA1: c.*+46C>A]. CONCLUSIONS: This mutation is in the αRNAmin binding site, so a single nucleotide substitution in this region can decrease mRNA stability by potentially compromising the binding of α-complex protein to αRNAmin, favouring the decay of α-globin mRNA via erythroid cell-enriched endoribonuclease cleavage. In this case, it is a non-deletional α-thalassaemia. However, in silico and empirical studies predicted that it could be a silent polymorphism. Functional studies should be carried out to confirm whether it is a pathological mutation or a silent polymorphism.

Hb Palencia: a novel δßδ-type two-way fusion variant with ß-globin-like expression levels.

AIMS: Fusion proteins of unequal recombination events at the ß-globin locus have pathological effect. The haemoglobin (Hb) variants of type Lepore are fusion proteins characterised by ß-like globin chains with a δ-globin (HBD) N-terminus and a ß-globin (HBB) C-terminus, whereas reciprocal products of underlying crossover events hold a HBB N-terminus and HBD C-terminus instead. Finally, Hb Parchman contains a ß-like globin chain with a central HBB fragment and HBD-derived N-termini and C-termini, whereas reciprocal hybrid proteins are as yet unknown. METHODS: The propositus was an 80-year-old Caucasian man, whose HbA1c quantification by HPLC (Variant II turbo) for exclusion of type-2 diabetes revealed an abnormal peak. Haemoglobins were analysed by ion-exchange HPLC (Variant II) and capillary electrophoresis (Sebia Capillarys Flex) and DNA by automatic Sanger sequencing of δ-globin and ß-globin genes. RESULTS: Sequencing showed an HBB-HBD-HBB hybrid gene, with HBD-derived central codons 9-31, and HBB-derived UTRs and complementary coding regions. The corresponding new hybrid haemoglobin (Hb Palencia) is represented at ≈40%, similar to HbA. CONCLUSION: Hb Palencia contains the first globin variant with internal HBD sequences and HBB-derived N-terminal and C-terminal and regulatory sequences. Relative quantity of the new ßδß-type variant suggests transcriptional control by HBB elements and a half-life similar to normal HBB.

Presence of acute and chronic renal failure in patients with paroxysmal nocturnal hemoglobinuria: results of a retrospective analysis from the Spanish PNH Registry.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening blood disease. With the advent of eculizumab treatment, renal function has substantially improved, although no data from real-world clinical practice are available. An observational, retrospective, multicenter study was conducted in Spain on clinical data obtained from outpatient visits of patients with PNH (Spanish PNH Registry) who had experienced acute (ARF) or chronic (CRF) renal failure. Of the 128 patients registered (April 2014), 60 were diagnosed with classic PNH. Twenty-seven (45.0%) patients with a mean age of 48.5 (±16.2) years had renal failure, ARF or CRF, and were included in this study. Near half of the patients (n = 13; 48.1%) presented with ARF alone, 33.3% (n = 9) had CRF with episodes of ARF, while 18.5% (n = 5) were diagnosed with CRF alone. For patients with diagnosis of PNH and renal failure (n = 27), the median time to the first ARF episode was 6.5 (CI 95%; 2.2, 14.9) years, whereas the median to the diagnosis of CRF was 14.5 (CI 95%; 3.8, 19.2) years after the diagnosis of PNH. Patients with ARF (n = 22) were treated with eculizumab and did not experience new episodes of ARF, except for one patient with sepsis. Of the patients with CRF, two received treatment without experiencing further episodes of ARF. Sixteen patients who completed treatment (11 with ARF and 5 with ARF + CRF) recovered from the episode of ARF or from CRF. Of the remaining patients treated with eculizumab, one patient improved from stages III to II, three patients stabilized without showing disease progression, and one patient progressed from stages III to IV. Treatment with eculizumab in PNH patients has beneficial effects on renal function, preventing ARF and progression to CRF.

Haemoglobinopathies that occur with decreased HbA2 levels: a gene mutation set involving the δ gene at a Spanish centre.

AIMS: Haemoglobin A2 (HbA2) consists of two globin chains, α and ß. Alterations in any of these genes influences the level of HbA2. Here, we present cases of structural Hb variants and thalassaemias which present either alone or together and reduce the level of HbA2 at varying degrees. Furthermore, we present a novel structural mutation in the δ globin gene, called Hb A2-Madrid. METHODS: The levels of HbA2 and HbF and the different haemoglobin variants were measured and analysed by ion exchange high performance liquid chromatography (HPLC, VARIANT II), the types of haemoglobins were determined by capillary zone electrophoresis (CZE) (Sebia) and the globin chains were determined by reversed-phase HPLC. Genetic analysis was performed by automatic sequencing of the α and δ genes as well as by multiple PCRs for the α globin genes. RESULTS: In α thalassaemia (n=94), the HbA2 levels ranged from 1.39% to 2.43%. Among individuals with δ thalassaemia (n=5), the HbA2 level of those with δ+ thalassaemia was 1.77%, and that of those with δ0 thalassaemia was 1.70%. Among the individuals with 뫧 thalassaemia (n=13), those who were homozygous lacked HbA2. All structural haemoglobinopathies (n=97) were heterozygous; the α chain variants (n=84) presented with an HbA2 level of 1.76%, while the δ chain variants (n=13) presented with a level of 1.75%. CONCLUSION: HbA2 is an essential parameter in the diagnostics of haemoglobinopathies. HPLC-EC and CZE allow the quantification of HbA2. Here, we show that quantification of HbA2 is critical for the identification of α, δ and ßδ thalassaemias. Structural variants are discovered by HPLC. Molecular genetics is required for the proper identification of the mutations. Only with this knowledge is genetic counselling possible.

Etiopathological mechanisms and clinical characteristics of hyperhemolysis syndrome in Spanish patients with thalassemia.

Hyperhemolysis syndrome (HHS) is characterized by severe intravascular hemolysis with a decrease in the reticulocyte count, which is triggered and aggravated by transfusion and cannot be explained by standard immunohematological studies. A nationwide study was conducted in order to retrospectively identify thalassemia patients with HHS in Spain in order to assess pre-disposing mechanisms for this syndrome. For this, the expression of adhesion (CD49, CD36) and complement-related molecules (C3a, CD59) and the levels of reticulocyte apoptosis and macrophage activation were measured in 4 thalassemia patients with HHS, 14 patients without HHS, and 10 healthy subjects. Five of the six thalassemia patients had δß-thalassemia. The patients were not alloimmunized prior to the syndrome, which was developed after the first transfusion in all but one case. Patients with δß-thalassemia did not respond to corticoids or immunoglobulins; only splenectomy was successful. The expression of CD49 (α4ß1 integrin) was far higher in patients who had experienced HHS (85.07 ± 18.46 vs. 46.28 ± 24.31; p < 0.01), and the difference remained significant after correcting by the number of molecules analyzed (Bonferroni p < 0.05). In our population, δß-thalassemia was the most common hemoglobinopathy in patients with HHS. Furthermore, the risk to develop this syndrome may be associated with an increased expression of α4ß1 integrin.

The utility of multiparametric seven-color flow cytometry in the detection of double hit lymphoma in ascitic fluid samples.

Double-hit lymphoma (DHL) is a rare type of lymphoma with concurrent chromosomal translocations of C-MYC with BCL2 or BCL6, associated with unfavorable prognosis. We describe a case of DHL in a 79-year-old female patient previously diagnosed with diffuse large B-cell lymphoma (DLBCL) with an early relapse in the ascitic fluid. A seven-color multiparametric flow cytometry immunophenotyping study of the ascitic fluid was carried out, and revealed 99.78% of large in size and high cellular complexity B-cells positive for CD19, CD10 (64.27%), CD45 dim, CD22 dim, CD25 (60%), CD43 bright, CD38 bright, and IgM (18.53%); and negative for CD20, CD5, CD23, CD79b, CD103, CD200, CD11c, and FMC7, and 78.99% without light chain expression and 21% with Lambda chain restriction. Due to the expression of CD19 and CD10 with overexpression of BCL-2 protein and due to CD43 and CD38 positivity detected, those cells showed features between DLBCL and Burkitt lymphoma. Fluorescence in situ hybridization (FISH) confirmed both c-MYC/IGH and BCL2/IGH rearrangement. Our findings may help to identify cases requiring additional cytogenetic analysis. © 2015 International Clinical Cytometry Society.

Identification of a novel mutation in the ß-globin gene 3' untranslated region (HBB: c.*+118A > G) in Spain.

The 3' untranslated region (3'UTR) region is well known to be associated with mRNA stability because of its associations with 3' end processing, polyadenylation and mRNA capping. Mutations located in this area cause a ß-thalassemia (ß-thal) phenotype compatible with ß(+)-thal. Two brothers, 49- and 41-years-old, were diagnosed with ß-thal intermedia (ß-TI) at 2 years of age. The ß-globin gene from the promoter region to the 3'UTR was sequenced and both brothers were diagnosed to be compound heterozygotes for the 3'UTR +1592 (A > G) (HBB: c.*+118A > G) and codon 39 (C > T) (HBB: c.118C > T) mutations. Their mother was a carrier of the nonsense codon 39 mutation and her hematological data suggested ß-thal trait; their father was a carrier of the 3'UTR +1592 mutation, though he did not have hematological parameters associated with ß-thal. The adenine at position +1592 or +118 bases downstream of the termination codon is highly conserved among primates and placental mammals, as it is located between the polyadenylation A signal (PAS) and the polyadenylation A cleavage (PAC) sites. Given its location, it is likely that this mutation would interfere with mRNA maturation; however, the clinical data of the heterozygous carriers show virtually no significant alterations. Therefore, we suggest that the impact on cleavage-stimulation factor (CstF) recognition of the mRNA sequence would be minimal and not significantly alter polyadenylation. Although the mechanism is not known, and because the carrier has no ß-thal minor, the mRNA is stable enough that the synthesis of the ß-globin chain is unaffected.

[Delta°-thalassemia by insertion of 27 base pairs in δ-globin gene with decreased hemoglobin A2 levels]. / Delta(0)-talasemia por inserción de 27 pares de bases en el gen δ-globina con descenso de los valores de hemoglobina A2.

BACKGROUND AND OBJECTIVE: We describe a novel delta-thalassemia mutation causing decreased hemoglobin (Hb) A2 levels associated with Hb Watts, variant Hb resulting from a trinucleotide deletion in Spain. PATIENTS AND METHOD: Hb variant analysis was performed by cation-exchange high performance liquid chromatography (HPLC) and capillary zone electrophoresis. Polymerase chain reaction and DNA sequence analyses were used to identify mutations in the δ- and α-globin genes. RESULTS: Abnormal Hb was observed on capillary zone electrophoresis in Z6 and by cation-exchange HPLC a slower peak than HbA was observed at an retention time of 4.19min. This variant Hb is called Hb Watts [α2 74(EF3)Asp->0 or α2 75(EF4)Asp->0; HBA2:c.226_228delGAC]. The decreased HbA2 percentage owes to an insertion of 27nt between nt 83 and 84 of IVS-I of the δ-globin gene. CONCLUSIONS: When analyzing a chromatogram, the possibility of the existence of delta-thalassemia or an HbA2 variant should be considered, apart from alfa-, beta-thalassemia and structural haemoglobinopathies. To this end, each of the peaks and their percentages should be considered to allow for correct interpretation and to avoid misdiagnosis as much as possible.

Caracterización molecular de dos nuevas mutaciones de α ° talasemia en 2 familias españolas (mutación - - ED y - - GP) / Molecular characterization of two new mutations of α ° thalassemia in two Spanish families (mutation -- ED and -- GP)

Fundamento y objetivo: La hemoglobina es un tetrámero constituido por dos cadenas α y dos cadenas β. Los dos genes estructurales que codifican la cadena α se encuentran localizados en el brazo corto del cromosoma 16. Los individuos normales tienen 4 genes α (αα/αα). Las α talasemias se producen generalmente por la deleción de uno, dos, tres o cuatro de los genes α. La deleción de ambos genes α dentro del mismo cromosoma (α° talasemia) se observa en individuos del área mediterránea y en el sudeste asiático. Material y método: Se estudian dos familias naturales de Madrid con anemia microcítica e hipocroma. El ADN extraído de los leucocitos de sangre periférica se digiere con diferentes enzimas de restricción e hibridación con sondas del cluster de genes α. Los extremos de la deleción se han caracterizado combinando las técnicas de Southern blot, reacción en cadena de la polimerasa (PCR) e hibridación in situ fluorescente (FISH). Resultados: En este trabajo presentamos dos nuevas mutaciones de α° talasemia en dos familias españolas, no descritas previamente en la bibliografía. La mutación (--ED) presenta una deleción de alrededor de 80kb con el punto de rotura 5’ en la coordenada +100 (± 3kb), mientras que el extremo 3’ HVR se sitúa en la coordenada 178±750bp. La segunda mutación (--GP) es más extensa, con pérdida de 145kb, situándose la deleción en la región 5’, entre las coordenadas 34 y 37, respetando el telómero. En la región centromérica la rotura se sitúa también en la coordenada 178±1,4kb. En ambas mutaciones se pierden los dos genes α, el gen θ y la región reguladora HS40. Conclusiones: La exacta identificación de estas mutaciones es esencial para determinar la función de los genes α en caso de consejo genético (AU)

Background and objetives: The two structural genes encoding the human α-globin chains are located on the short arm of chromosome 16. Normal individuals have four genes α (αα/αα). α-thalassemias are usually produced by the deletion of one, two, three, or four α genes. Deletion of both α genes within the same chromosome (α° thalassemia) is commonly observed in individuals from the Mediterranean basin and Southeast Asia. Material and methods: We study two natural families of Madrid with microcytic hypochromic anemia. The DNA extracted from peripheral blood leukocytes was digested with different restriction enzymes and hybridization with probes of gene cluster α. The ends of the deletion were characterized by combining the techniques of Southern blot, PCR and FISH. Results: We present two new mutations of α° thalassemia in two Spanish families, not previously described in the literature. The deletion (--ED) is ∼80kb with the point of bread 5’ in the coordinate +100 (± 3kb), whereas the end 3’HVR places in the coordinate 178±750bp. The second deletion (--GP) is more extensive, with loss of 145kb, placing the deletion in the end 5’ between the coordinates 34 and 37, respecting therefore the telomere. In the centromeric region the point of break places as the previous one in the coordinate 178±1.4bp. Conclusions: In both mutations both alpha genes were deleted, the gene θ and the region HS40. The exact identification of these deletions is essential to determine the function of the genes α with a view to a possible genetic diagnosis (AU)

[Prenatal diagnosis of hemoglobinopathies and thalassemias]. / Diagnóstico prenatal de hemoglobinopatías y talasemias.

BACKGROUND AND OBJECTIVE: Thalassemias are the most common single-gene disorders in the world population and the most important health problem in several countries. The best program of prevention of new births is prenatal diagnosis. Here we gather the experience from 1996 of the Spanish Group of Erythropathology related to the prenatal diagnosis of hemoglobinopathies in Spain. SUBJECT AND METHOD: 36 couples carriers of beta-thalassemia or Hb S were studied. Fetal material was obtained by amniocentesis and BCV. The genotype was determined by molecular biology technologies. RESULTS: We observed 3 spontaneous abortions (8.3%), 6 interruptions of pregnancy (16.7%) and a case of maternal contamination (2.8%). Prenatal diagnosis could be completed in 97.2% of the cases (35). CONCLUSIONS: In our experience, with regards to hemoglobinopathies, it is essential to provide a good genetic advice in order to identify the molecular alteration in the progenitors before the first pregnancy. This would allow a prenatal diagnosis during the first quarter and, in case of a positive result, to perform an early interruption of the pregnancy without risks.

[Hemoglobin Stanleyville II [alpha78(EF7)Asn --> Lys]. First case described in Spain]. / Hemoglobina Stanleyville II [ alpha78(EF7)Asn --> Lys]. Primer caso descrito en España.

BACKGROUND AND OBJECTIVE: Structural hemoglobinopathies are the result of mutations in the genes of globin, which determine a qualitative alteration in the expression of these genes. Most alterations do not originate any significant change, and correspond to silent or asymptomatic forms. This study proves a new case of hemoglobin (Hb) Stanleyville II. PATIENTS AND METHOD: The propositus was a 72 years old Caucasian woman, from the Canary Islands. Her hematological data were: Hb 14.3 g/dl; hematocrit 44.4%; mean corpuscular volume 85.8 fl; mean corpuscular hemoglobin 27.7 pg; red cell distribution width 15.1%; reticulocytes 1.2%; HbA2 3.1% and HbF 1.6%. Electrophoretic studies in cellulose acetate electrophoresis at alkaline pH = 8.6 and isoelectrofocusing showed an anomalous Hb similar to HbS. The anomalous Hb did not appear in agar citrate electrophoresis (pH 6.0). The analysis by reverse phase high performance liquid chromatography for globin chains showed an X anomalous after A. RESULTS: Molecular analysis by sequentiation of the polymerase chain reaction products genes 1 and 2 showed the mutation AAC --> AAA at CD78 of second gene 2 in heterozygote state, which leads the change of asparagine to lysine. CONCLUSIONS: The substitution of an amino acid with neutral charge like asparagine for another one with positive charge like lysine in the segment EF, which corresponds to the external surface of the tertiary structure of the chain of globin, determines the change of charge in the chain. This allows an easy differentiation by electrophoretic and chromatographic methods. Nevertheless, owing to its position in the chain, which is not critique for the stability, solubility and affinity for the oxygen allows for silent or asymptomatic forms. The Hb Stanleyville II had been described before in black families of the Congo, Uganda, USA, Alsace and Brazil. This case represents the first case described in Spain.

Origin of the frameshift codons 41/42 (-TCTT) mutation in the first cases described in the Spanish population.

Thalassemias are hereditary anemias. In beta-thalassemia (beta-thal), beta-globin synthesis is either deficient or absent. A high incidence of beta-thal is found in populations of Mediterranean and African origin. Smaller, but significant concentrations of beta-thal are present throughout the Middle East, India, Pakistan and China, while sporadic cases have been reported in most ethnic groups. Over 200 beta-thal mutations have been described so far. But each population group displays its own mutations. In Spain, as in other countries of the Mediterranean region, the most often seen mutations are codon 39 (C > T); IVS-I-1 (G > A); IVS-I-6 (T > C) and IVS-I-110 (G > A). However, a large number of rarer alleles have been observed both in Spain and other populations. The frameshift codons (FSC) 41/CD42 (-TCTT) mutation is a rather common allele in individuals of Chinese origin, but rare in the Mediterranean region, although, it has been recorded in East Asian populations. We describe the first eight Spanish patients displaying the FSC 41/42 mutation. This mutation was initially detected with a real-time polymerase chain reaction (PCR) method on a LightCycle, using a probe designed to detect mutations in codons 37 and 39, and subsequently specifically characterized by automatic sequencing. The haplotype found in our patients suggested that this mutation has not arisen independently in our population but must be taken into account when identifying most beta-thal mutations.

Frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in a cohort of 1,000 neonates in Madrid (Spain).

For centuries in Europe, population movements have contributed to ethnic groups, cultures, and consequently, inheritance mixing. There are certain genetic diseases such as hereditary hemochromatosis whose distribution is directly related to the population movements. The objective of the present investigation was to determine the C282Y and H63D mutation frequency of the HFE gene in a cohort study of 1,000 neonates in the Community of Madrid (Spain), thus contributing to the HFE gene mutations distribution research in Europe and establishing the origin of the mutations in Spain. The allelic frequency of C282Y mutation was 1.7% (CI 95% 1.1-2.3) and the H63D allele was present in 16.4% of chromosomes (CI 95% 14.8-18). In Spain, the presence of C282Y mutation and its distribution could be due more to Celtic than to Viking legacy, whereas it is assumed that the one in relation to the H63D variant occurred in the Basque Country during the Paleolithic Period.